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exposing the UCP3-ko mice to a series of conditions pre- viously related to induction of uncoupling proteins (high-fat feeding, cold exposure, stimulation with thyroid hormone) did not result in differences between the UCP3-ko and their wild-type littermates (22,23), which was anticipated given the previous findings in mice overexpressing UCP3 (15). Interestingly however, Vidal-Puig et al. reported increased production of the superoxide anion in vitro and augmented mitochondrial aconitase production, indicating in vivo pro- duction of reactive oxygen species (ROS) in the UCP3-ko mice (23). These findings led the authors to suggest that one of the functions of UCP3 could be the prevention of exces- sive oxidative stress by lowering the mitochondrial mem- brane potential, thus lowering the probability for electrons to interact with oxygen (23). to induce uncoupling, both superoxides and coenzymeQ are obligatory factors (18). Indeed, it was shown that exogenous superoxide induced uncoupling in skeletal muscle mito- chondria only in the presence of coenzymeQ, showing a 2-fold increase in uncoupling if UCP3 levels were doubled by fasting and a lack of effect of superoxide in mitochondria isolated from UCP3-ko mice (18). The increased uncou- pling was inhibited by purine nucleotides, and the activation of uncoupling by superoxide was abolished if 0.3% bovine serum albumin was added to the medium and was restored by adding palmitic acid in the micromolar range (300 indicate that UCP3 can be involved in regulation of oxida- tive stress, one should keep in mind that the findings re- ported are derived from isolated mitochondria exposed to high levels of exogenous superoxide, which complicates extrapolation of these findings to intact humans. With re- spect to this, it is of relevance to note that the obligatory role of coenzymeQ in inducing uncoupling has recently been under debate; it was shown that after reconstitution of UCP1, 2, and 3 in liposomes, the fatty acid catalyzed proton flux in these liposomes was unaffected by the presence of coenzymeQ (24). Rather, it was shown that the previously observed relevance of coenzymeQ in uncoupling was com- pletely accounted for by the effect of its solvent dichlo- romethane (24). coupling in UCP3-ko mice. This approach revealed that the unidirectional rates of ATP synthesis were increased in UCP3-ko mice, whereas tricarboxylic acid (TCA) cycle flux remained unaltered (25). These findings were interpreted as evidence for uncoupling activity by UCP3 in vivo (25). It should be recognized, however, that increased ATP synthe- interpret, and simply interpreting it as increased coupling seems premature. Affecting UCP3 that we reported significant correlations of UCP3 mRNA with both BMI (negative) and resting metabolic rate (posi- tive) (27), suggesting that skeletal muscle UCP3 content is related to energy expenditure. In this context, it was already recognized that hyperthyroidism, characterized by increased energy expenditure, was associated with increased proton leak in liver mitochondria, whereas the opposite was ob- served in hypothyroidism (28). Interestingly, hypothyroid rats had only 32% of the UCP3 mRNA levels found in controls. Treatment of hypothyroid, but also euthyroid, rats with thyroid hormone induced a 4.7- and 6.2-fold increase in skeletal muscle UCP3 mRNA (8). These findings were extended with assessments of mitochondrial respiration rates in the transition from the hypothyroid to the euthyroid state, showing that state 3 and state 4 respiration rates were ranked hierarchically from hypothyroid ating the proton leak observed in hyperthyroidism. More compelling data for a role of UCP3 in thyroid hormone- induced increases in energy expenditure are presented by de Lange et al. (30), showing that after a single dose injection of thyroid hormone, UCP3 mRNA peaked at 24 hours after injection. The increase in mRNA was reflected in increased protein levels 65 hours after injection, with a concerted increase in resting metabolic rate assessed in vivo (30). concerted up-regulation of UCP3 at the protein level (2.8- fold) (31). flux without an effect on unidirectional ATP synthesis rate; this was interpreted by the authors as a 60% decrease in mitochondrial coupling and has led them to suggest that UCP3 is responsible for the increased energy expenditure and thermogenesis observed after administration of thyroid hormone (31). In humans treated with thyroid hormone, similar changes in TCA cycle flux and unidirectional ATP synthesis rate were observed (32). Unfortunately, no UCP3 levels were reported in that study (32). It should, however, be noted that, although very elegant and noninvasive, results from creased unidirectional ATP synthesis rate without effect on TCA cycle flux has been interpreted as decreased uncou- pling (25), and, at the same time, increased TCA cycle flux without an effect on ATP synthesis rate has been taken as a |
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